As the demand of energy increases worldwide, fossil fuel is rapidly depleted. Therefore, alternative sources of energy have to be evaluated to meet the global energy demand. Methane, hydrogen and ethanol are considered as potential substitutes for fossil fuels. Among these three candidates, ethanol is commonly considered to be a good choice for an alternative liquid fuel in the near term.
The process of ethanol production using biomass as a feedstock is well known (http://www.vermontbiofuels.org/biofuels/ethanol.shtml). In this process, both glucose and pentose are fermented to ethanol by a microorganism. Currently, yeast (Saccharomyces cerevisiae) is often used in the process, see, Almeida, J. R. M., et al., J. of chem. tech. and biotech., 2007, 82(4): p. 340-349.
When choosing a microorganism for fermentation, several important traits may be considered, including yield, ethanol tolerance, productivity, and growth requirements, see, Dien, B. S., et al., Applied microbial biotechnology, 2003, 63: p. 258-266. Among these traits, ethanol yield has received much attention because feedstock may account for greater than one-third of the production costs. If ethanol yield is high, less feedstock would be needed to produce the same amount of ethanol. Consequently, the production cost could be reduced, so high ethanol yield is often important. Based on this requirement, Zymomonas mobilis, which was found to have the highest ethanol yield on sugar complex containing glucose, see, Lee, K. J., et al., Biotechnology letters, 1980. 2(11): p. 487-492; Rogers, P. L., et al., Process Biochemistr, 1980, 15(6): p. 7-11; and Rogers, P. L., et al., Adv. Biotechnol., [Proc. Int. Ferment. Symp.] 6th, 1980, became one of the most promising microorganisms having the potential to replace the yeast for ethanol production. This microorganism has been demonstrated to have ethanol yields up to 97% of the theoretical value. When compared with traditional yeast fermentation, it could achieve 5 to 10% higher yield, see, El-Mansi, M., Fermentation microbiology and biotechnology, 2007: CRC Press; and Fraser-reid, B., et al., Glycosience: Chemistry and Chemical Biology. 2001: Springer. Another advantage of Z. mobilis is its high ethanol productivity. The volumetric ethanol productivity of Z. mobilis could be five-fold higher than S. cerevisiae. Additional advantages of Z. mobilis for ethanol production are reported by Rogers, P. L., et al., in Biotechnology letter, 1979, 1: p. 165-170, and include the high sugar tolerance, the low production cost and the ability to ferment sugar at low pH. Z. mobilis could grow at high concentrations of glucose (10-25%). This microorganism is also acid tolerant and could grow over a pH range of 3.5 to 7.5. So the fermentations are generally resistant to bacterial contamination.
Although Z. mobilis is better than yeast in some aspects, it has not been used commercially for a number of reasons. First, Z. mobilis typically only uses glucose, fructose and sucrose as their substrates. Since pentoses such as xylose is a major component of hemicellulose in most biomass feedstock, it is usually essential for a fermenting microorganism to use this sugar in ethanol production for a good product yield from biomass. Fortunately metabolic engineering has been successfully applied to develop a Zymomonas strain to ferment xylose, (see, Zhang, M., Engineering Zymomonas mobilis for efficient ethanol production from lignocellulosic feedstocks. ACS national meeting, 2003 and U.S. Pat. No. 7,223,575, which is incorporated herein by reference to the extent that it is not inconsistent) and as well as arabinose, see, Mohagheghi, A., et al., Applied biochemistry and biotechnology, 2002, 98-100: p. 885-898. By genetic engineering technology, engineered Z. mobilis could potentially use all sugars present in most biomass feedstock. Secondly, Z. mobilis is sensitive to various inhibitors, including ethanol, aliphatic acids, such as acetic acid, formic acid; furan derivatives, such as 2-furaldehyde, 2-furoic acid; and phenolic compounds, such as vanillin and hydroxybenzoic acid, founds in the biomass, see, Lawford, H. G., et al., Applied biochemistry and biotechnology, 1993, 39/40: p. 687-699. As reported by Jeon, Y. J., et al., Biotechnology letters, 2002, 25: p. 819-824, the toxicity of acetic acid intensified during xylose fermentation. The pretreated biomass by dilute-acid usually contains up to 1.5% acetic acid (w/v) due to the hydrolysis of the acetylated pentoses in hemicellulose. Before using Z. mobilis in industry, this inhibition problem has to be addressed.
Several researchers have tried to develop acetic acid tolerant strains Z. mobilis by genetic modification. Among them, Rogers et al. used N-methyl N′-nitro-N-nitrosoguanidine (NTG) treatment in 1998 to develop several strains of Z. mobilis. Baumler et al., in Applied biochemistry and biotechnology, 2006, 134: p. 15-26, proposed recombinant DNA technology to enhance the acid tolerance in Z. mobilis (CP4). Among other methods that have been tried to address the acetic acid toxicity of Z. mobilis include, optimizing the fermentation conditions by removal of acetic acid from pretreated biomass by ion-exchange resins and ion exchange membranes (see, Han, B., et al., Desalination, 2006, 193: p. 361-366) and finding optimum fermentation conditions for the recombinant Z. mobilis. 
Moreover, even though many modification methods are known, (see, Foster, P. L., Annual review of genetics, 1999, 33: p. 57-88; Foster, P. L., Annual reviews of microbiology, 1993, 47: p. 467-504 and Rosenberg, S. M., Evolving responsively: Adaptive mutation. Nature Reviews Genetics, 2001. 2(7): p. 504-515), nobody has successfully modified Z. mobilis to develop inhibitor tolerance and/or pentose consumption in a cost-efficient manner. Accordingly, there remains a continuing need to develop more inhibitor tolerant (such as acetic acid tolerant) strains of Z. mobilis that can be used for ethanol production from biomass. There also remains a continuing need to develop a strain more capable of fermenting pentoses.
Provided herein are Zymomonas mobilis mutant strains that are more tolerant to various inhibitors sometimes found in biomass and/or that may ferment additional carbohydrates, methods of obtaining the mutant strains, and methods of using the mutant strains to prepare ethanol from biomass.
In one embodiment the invention pertains to processes for adaptively mutating a bacteria such as one from the genus Zymomonas. The process of adaptively mutating the bacteria comprises sequentially culturing the bacteria in the presence of one or more selective pressures which are consecutively increased. Then, a mutant strain which is more adapted to the selective pressure is isolated.
In another embodiment, the invention pertains to making a Zymomonas mobilis strain more tolerant to an inhibitor. The process comprises first growing a Zymomonas mobilis strain in a medium substantially free of an inhibitor. Next, the Zymomonas mobilis strain is sequentially cultured in the presence of consecutively higher concentrations of the inhibitor. Then, a mutant strain adapted to a higher inhibitor concentration isolated.
In another embodiment, the invention pertains to a process for making a Zymomonas mobilis strain capable of increased carbohydrate fermentation of one or more carbohydrates selected from xylose, arabinose, mannose and mixtures thereof. The process comprises first growing a Zymomonas mobilis origin strain in a medium comprising glucose. Next, the Zymomonas mobilis strain is sequentially cultured in the presence of consecutively higher concentrations of one or more carbohydrates selected from xylose, arabinose, mannose and mixtures thereof and lower amounts of glucose. Then, a mutant strain capable of increased carbohydrate fermentation of one or more carbohydrate selected from xylose, arabinose, mannose and mixtures thereof is isolated.
In another embodiment, the mutant Z.mobilis strains made by the techniques of the present invention, e.g., acetic acid inhibitor tolerant Z.mobilis strains, often have a number of unique characteristics or combinations of unique characteristics. The non-naturally occurring, biologically pure Zymomonas mobilis mutant strain may be characterized by substantially exhibiting one or more of the following characteristics: (1) a lag phase of less than about one day, preferably less than 9 hours; or (2) a specific growth rate of at least about 0.15 h−1, preferably at least about 0.3 h−1 or (3) an ethanol yield of at least about 95% of theoretical yield; wherein the characteristics are exhibited while fermenting at a pH of about 6 in an RM medium with 50 g/L glucose and 1.6% acetic acid concentration. In some embodiments the strain substantially exhibits at least 2 or even all 3 of the aforementioned characteristics.
In another embodiment, the mutant Z.mobilis strains made by the techniques of the present invention, e.g., enhanced carbohydrate fermentation Z.mobilis strains, often have a number of unique characteristics or combinations of unique characteristics. The non-naturally occurring, biologically pure Zymomonas mobilis mutant strain may be characterized by substantially exhibiting one or more of the following characteristics: (1) an ethanol yield of at least about 85%, preferably at least about 90% of theoretical yield; or (2) a volumetric ethanol productivity of at least about 0.5, preferably at least about 0.8 grams of ethanol per liter of reactor per hour (g/l/h); or (3) a specific ethanol productivity of at least about 0.9, preferably at least about 0.95 grams per gram of dry cell mass per hour (g/g/h); or (4) a xylose consumption rate of at least about 1.8, preferably at least about 2.0 grams per gram of dry cell mass per hour (g/g/h); or (5) an ability to consume 5% (w/v) xylose in less than about 40 hours, preferably less than about 36 hours; wherein the characteristics are exhibited while fermenting in an RM medium with 50 g/L xylose without glucose.
In another embodiment, the invention pertains to a non-naturally occurring, biologically pure Zymomonas mobilis mutant strain characterized by substantially exhibiting one or more of the following characteristics:    (1) a lag phase of less than about one day; or    (2) a specific growth rate of at least about 0.15 h−1; or    (3) an ethanol yield of at least about 95% of the theoretical yield;    (4) an ethanol yield of at least about 85% of theoretical yield; or    (5) a volumetric ethanol productivity of at least about 0.5 grams of ethanol per liter of reactor per hour; or    (6) a specific ethanol productivity of at least about 0.9 grams of ethanol per gram of dry cell mass per hour; or    (7) a xylose consumption rate of at least about 1.8 grams of xylose per gram of dry cell mass per hour; or    (8) an ability to consume 5% (w/v) xylose in less than about 40 hours; wherein the one or more characteristics (1)-(3) are exhibited while fermenting at a pH of about 6 in an RM medium with 50 g/L glucose and 1.6% acetic acid concentration and wherein the one or more characteristics (4)-(8) are exhibited while fermenting in an RM medium with 50 g/L xylose without glucose.